Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

ABSTRACT

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps:
         a) optionally pre-treatment of the ligno-cellulosic material;   b) optionally washing of the optionally pre-treated ligno-cellulosic material;   c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61;   d) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and   e) optionally recovery of a fermentation product;
 
wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.

FIELD OF THE INVENTION

The invention relates to a process for the enzymatic hydrolysis oflignocellulosic material and fermentation of sugars.

BACKGROUND OF THE INVENTION

Ligno-cellulosic plant material, herein also called feedstock, is arenewable source of energy in the form of sugars that can be convertedinto valuable products e.g. sugars or bio-fuel, such as bio-ethanol.During this process, (ligno or hemi)-cellulose present in the feedstock,such as wheat straw, corn stover, rice hulls, etc., is converted intoreducing sugars by (hemi)-cellulolytic enzymes, which then areoptionally converted into valuable products such as ethanol bymicroorganisms like yeast, bacteria and fungi.

Since the (hemi)-cellulose is crystalline and entrapped in a network oflignin the conversion into reducing sugars is in general slow andincomplete. Typically, enzymatic hydrolysis of untreated feedstockyields sugars <20% of theoretical quantity. By applying a chemical andthermo-physical pre-treatment, the (hemi)-cellulose is more accessiblefor the (hemi)-cellulolytic enzymes, and thus conversions go faster andat higher yields.

A typical ethanol yield from glucose, derived from pre-treated cornstover, is 40 gallons of ethanol per 1000 kg of dry corn stover (Badger,P, Ethanol from cellulose: a general review, Trends in new crops and newuses, 2002, J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria, Va.)or 0.3 g ethanol per g feedstock. The maximum yield of ethanol oncellulose base is approximately 90%.

Cellulolytic enzymes—most of them are produced by species likeTrichoderma, Humicola and Aspergillus—are commercially used to convertpre-treated feedstock into a mash containing insoluble (hemi)cellulose,reducing sugars made thereof, and lignin. Thermostable cellulolyticenzymes derived from Rasamsonia, have been used for degradingligno-cellulosic feedstock and these enzymes are known for theirthermostability, see WO2007091231. The produced mash is used in afermentation during which the reducing sugars are converted into yeastbiomass (cells), carbon dioxide and ethanol. The ethanol produced inthis way is called bio-ethanol.

The common production of sugars from pre-treated ligno-celullosicfeedstock, the hydrolysis also called liquefaction, pre-saccharificationor saccharification, typically takes place during a process lasting 6 to168 hours (Kumar, S. , Chem. Eng. Technol. 32 (2009) 517-526) underelevated temperatures of 45 to 50° C. and non-sterile conditions. Duringthis hydrolysis, the cellulose present is partly (typically 30 to 95%,dependable on enzyme activity and hydrolysis conditions) converted intoreducing sugars. In case of inhibition of enzymes by compounds presentin the pre-treated feedstock and by released sugars; and to minimizethermal inactivation, this period of elevated temperature is minimizedas much as possible.

The fermentation following the hydrolysis takes place in a separatepreferably anaerobic process step, either in the same or in a differentvessel, in which temperature is adjusted to 30 to 33° C. (mesophilicprocess) to accommodate growth and ethanol production by microbialbiomass, commonly yeasts. During this fermentation process, theremaining (hemi) cellulosic material is converted into reducing sugarsby the enzymes already present from the hydrolysis step, while microbialbiomass and ethanol are produced. The fermentation is finished once(hemi) cellulosic material is converted into fermentable sugars and allfermentable sugars are converted into ethanol, carbon dioxide andmicrobial cells. This may take up to 6 days. In general the overallprocess time of hydrolysis and fermentation may amount up to 13 days.

The so obtained fermented mash consists of non-fermentable sugars,non-hydrolysable (hemi) cellulosic material, lignin, microbial cells(most common yeast cells), water, ethanol, dissolved carbon dioxide.During the successive steps, ethanol is distilled from the mash andfurther purified. The remaining solid suspension is dried and used as,for instance, burning fuel, fertilizer or cattle feed.

WO2010080407 suggests treating cellulosic material with a cellulasecomposition under anaerobic conditions. Removal or exclusion of reactiveoxygen species may improve the performance of cellulose-hydrolyzingenzyme systems. Hydrolysis of cellulosic material, e.g., lignocellulose,by an enzyme composition can be reduced by oxidative damage tocomponents of the enzyme composition and/or oxidation of the cellulosicmaterial by, for example, molecular oxygen.

WO2009046538 discloses a method for treating lignocellulosic feedstockplant materials to release fermentable sugars using an enzymatichydrolysis process for treating the materials performed under vacuum andproducing a sugar rich process stream comprising reduced amounts ofvolatile sugar/fermentation inhibiting compounds such as furfural andacetic acid. Apart from removing volatile inhibitory compounds, othercompounds and/or molecules that are also removed include nitrogen,oxygen, argon and carbon dioxide.

With each batch of feedstock, enzymes are added to maximize the yieldand rate of fermentable sugars released from the pre-treatedligno-cellulosic feedstock during the given process time. In general,costs for enzymes production, feedstock to ethanol yields andinvestments are major cost factors in the overall production costs(Kumar, S. Chem. Eng. Technol. 32 (2009) 517-526). Thus far, cost ofenzyme usage reduction is achieved by applying enzyme products from asingle or from multiple microbial sources (WO 2008/008793) with broaderand/or higher (specific) hydrolytic activity which use aims at a lowerenzyme need, faster conversion rates and/or a higher conversion yields,and thus at overall lower bio-ethanol production costs. This requireslarge investments in research and development of these enzyme products.In case of an enzyme product composed of enzymes from multiple microbialsources, large capital investments are needed for production of eachsingle enzyme compound.

It is therefore desirable to improve the above process involvinghydrolysis and fermentation.

SUMMARY OF THE INVENTION

An object of the invention is therefore to provide a process in whichthe hydrolysis step is conducted at improved conditions. Another objectof the invention is to provide a process involving hydrolysis having areduced process time. Further object of the invention is to provide aprocess, wherein the dosage of enzyme may be reduced and at the sametime output of useful hydrolysis product is maintained at the same levelor even increased. Another object is to provide a process involvinghydrolysis, wherein the process conditions of the hydrolysis areoptimized. A still further object of the invention is to provide aprocess involving hydrolysis, wherein the output of useful hydrolysisproduct is increased using the same enzyme dosage. One or more of theseobjects are attained according to the invention.

The present invention provides a process for the preparation of a sugarproduct from ligno-cellulosic material, comprising the following steps:

-   -   a) optionally pre-treatment of the ligno-cellulosic material;    -   b) optionally washing of the optionally pre-treated        ligno-cellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pre-treated ligno-cellulosic material using an enzyme        composition comprising at least two cellulase and whereby the        enzyme composition at least comprises GH61; and    -   d) optionally recovery of a sugar product;        wherein after the pre-treatment and before and/or during the        enzymatic hydrolysis oxygen is added to the ligno-cellulosic        material.

Furthermore the present invention provides a process for the preparationof a fermentation product from ligno-cellulosic material, comprising thefollowing steps:

-   -   a) optionally pre-treatment of the ligno-cellulosic material;    -   b) optionally washing of the optionally pre-treated        ligno-cellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pre-treated ligno-cellulosic material using an enzyme        composition comprising at least two cellulase and whereby the        enzyme composition at least comprises GH61;    -   d) fermentation of the hydrolysed ligno-cellulosic material to        produce a fermentation product; and    -   e) optionally recovery of a fermentation product;        wherein after the pre-treatment and before and/or during the        enzymatic hydrolysis oxygen is added to the ligno-cellulosic        material.

Preferably the oxygen is added during the enzymatic hydrolysis step c).

In a preferred embodiment the oxygen is added in the form of (gaseous)bubbles.

Surprisingly, according to the invention, by the addition of oxygen itis possible to attain many process advantages, including optimaltemperature conditions, reduced process time, reduced dosage of enzyme,re-use of enzymes, higher yields and other process optimizations,resulting in reduced costs.

In one embodiment of this process, the fermentation time is 5 to 120hours. In an embodiment the stable enzyme composition used retainsactivity for 30 hours or more. According to a further embodiment thehydrolysis is preferably conducted at a temperature of 45° C. or more,more preferably at a temperature of 50° C. or more and still morepreferably at a temperature of 55° C. or more. In a preferredembodiment, the enzyme composition is derived from a fungus, preferablya microorganism of the genus Rasamsonia or the enzyme compositioncomprises a fungal enzyme, preferably a Rasamsonia enzyme. The processof the invention will be illustrated in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose (g/l)released by the TEC-210 mix (1), 4E-GH61 mix (2) and 4E-EG mix (3).

FIG. 2: The enzymatic hydrolysis in a 270 liter reactor (pilot plantscale) whereby the glucan conversion (%) is shown in 20% aCS as functionof the process time (hours) for 3.75 mg TEC210/g feedstock DM for low DO(—□—) and high DO (—▪—).

FIG. 3: The effect of the dissolved oxygen concentration (DO) on glcanhydrolysis in pretreated lignocellulosic feedstock as function ofhydrolysis time for 2.5mg/g of enzyme and DO=0.030 mol/m³ (—♦—) and3.5mg/g of enzyme and DO=0.005 mol/m³ (—▪—).

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element.

In the context of the present invention “improved”, “increased”,“reduced” is used to indicate that the present invention shows anadvantage compared to the same situation, process or process conditionsexcept that no extra oxygen is added. Within the context of the presentinvention “measured under the same conditions” or “analysed under thesame conditions” etc. means that the process of the invention and thesame process without addition of oxygen are performed under the sameconditions (except the oxygen addition) and that the results of thepresent process, if compared to the process without oxygen addition, aremeasured using the same conditions, preferably by using the same assayand/or methodology, more preferably within the same or parallelexperiment. Conditions of the hydrolysis are an example of suchconditions.

In prior art it is suggested to improve the hydrolysis of cellulolyticmaterial by using anaerobic (WO2010/080407) or vacuum (WO2009/046538)conditions during the enzymatic hydrolysis. In the processes of bothdocuments the oxygen level was decreased. It has now surprisingly beenfound that the hydrolysis of the present invention shows results in animproved reaction product that gives higher amounts of (reduced) sugarproducts and/or desired fermentation products in the fermentationfollowing the hydrolysis as compared to a process wherein no oxygen isadded. In general an increase of the glucose conversion is observed of 5to 15 w/w %.

Oxygen can be added in several ways. For example oxygen can be added asoxygen gas, oxygen enriched gas such as oxygen enriched air or air.Oxygen can be added continuously or dis-continuously. By oxygen “isadded” is meant that oxygen is added to the liquid phase (comprising thelingo-cellulosic material) in the hydrolysis reactor and not that oxygenis present in the headspace in the reactor above the liquid phasewhereby the oxygen has to diffuse from the headspace to the liquidphase. So preferably the oxygen is added as bubbles, most preferably assmall bubbles.

In case the enzyme may be damaged by the presence or addition of oxygen,milder oxygen supply may be used. In that case a balance can be foundbetween the improved glucose production and the enzyme performance. Theaddition of the oxygen to the cellulolytic material can be done beforeand/or during the enzymatic hydrolysis. In case oxygen is added ingaseous form, oxygen-containing gas can be introduced, for exampleblown, into the liquid hydrolysis reactor contents of cellulolyticmaterial. In another embodiment of invention the oxygen-containing gasis introduced into the liquid cellulolytic material stream that willenter the hydrolysis reactor. In still another embodiment of theinvention the oxygen containing gas is introduced together with thecellulolytic material that enters the hydrolysis reactor or with part ofthe liquid reactor contents that passes an external loop of the reactor.In most cases the addition of oxygen before entering the hydrolysisreactor is not sufficient enough and oxygen addition may be done duringthe hydrolysis as well. In another embodiment of the invention thegaseous phase present in the upper part of the reactor (head space) iscontinuously or dis-continuously refreshed with the oxygen-containinggas. In the latter case (vigorous) mixing or stirring is needed to getthe oxygen as bubbles and/or by diffusion into the liquid reactorcontents preferably in combination with over-pressure in the reactor. Ingeneral flushing the headspace with air in combination with (vigorous)mixing or stirring may introduce sufficient oxygen into the cellulosicmaterial in the hydrolysis reactor for reactors up to a size of 100liter to 1 m³. At larger scale, for example in a reactor of 50 m³ ormore, for example 100 m³, so much energy is needed for vigorous stirringthat from economic point of view this will not be applied in acommercially operating process.

According to the present invention the oxygen may be added before thehydrolysis step, during part of the hydrolysis step, during the wholehydrolysis step or a combination of before or during the hydrolysisstep. Advantageously the oxygen is added during the first half in timeof the hydrolysis step. The addition of oxygen during only part of thehydrolysis may be done for example in case of oxidation damage of theenzyme(s) occurs. In case the oxygen present in the hydrolysis reactorcontents or the sugar product or the hydrolysate formed in thehydrolysis step might influence or disturb in the subsequentfermentation step, oxygen addition may be done except for the last partof the hydrolysis and thus (most of) the oxygen is consumed before thehydrolysed biomass enters the fermentation reactor.

Several examples of aeration during the enzymatic hydrolysis process aregiven in the Examples to show the beneficial effect of the presentinvention. This beneficial effect is found for several substrates orfeedstocks and therefore believed to be present for the hydrolysis ofall kind of substrates or feedstocks.

Several examples of enzyme compositions for the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for several enzymecompositions and therefore believed to be present for all kind ofhydrolysing enzyme compositions.

To a further preferred embodiment of the invention the oxygenconcentration in the liquid phase (DO), wherein the ligno-cellulosicmaterial is present during the enzymatic hydrolysis, is at least 0.001mol/m³, preferably at least 0.002 mol/m³, more preferably at least 0.003mol/m³ and even more preferably more than 0.01 mol/m³,for example morethan 0.02 mol/m³ or 0.03 mol/m³. In reactors of less than 1 m³ DO valuesof below 0.01 mol/m³ or 0.02 mol/m³ will be obtained by slow stirring.Vigorous mixing or stirring at such scale introduces part of the gasphase of the headspace into the reaction liquid. For example the mixingor stirring may create a whirlpool that draws oxygen into the liquid. Ingeneral flushing the headspace with air in combination with (vigorous)mixing or stirring will introduce sufficient oxygen into the cellulosicmaterial in the hydrolysis reactor for reactors up to a size of 100liter to 1 m³. At larger scale, for example in a reactor of 50 m³ ormore, for example 100 m³, so much energy is needed for vigorous stirringthat from economic point of view this will not be applied in acommercially operating process. In general in large reactors, stirringor mixing without introducing air or oxygen will result in DO values ofless than 0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygengeneration or production the oxygen concentration in the liquid phase(aeration or addition of oxygen), the oxygen concentration in the liquidphase wherein the ligno-cellulosic material is present during theenzymatic hydrolysis, is preferably at most 80% of the saturationconcentration of oxygen under the hydrolysis reaction conditions, morepreferably at most 0.12 mol/m³, still more preferably at most 0.09mol/m³, even more preferably at most 0.06 mol/m³, even still morepreferably at most 0.045 mol/m³ and most preferably at most 0.03 mol/m³.Temperature and pressure will influence the DO. The preferred andexemplary mol/m³ values given above relate to normal atmosphericpressure and a temperature of about 62° C. The skilled person in the artwill appreciate favourable DO values on basis of the present teachings.

The oxygen addition in the form of air or other oxygen-containing gasaccording to the invention may also be used to at least partially stiror mix the hydrolysis reactor contents.The present process of theinvention shows especially on pilot plant and industrial scaleadvantages. Preferably the hydrolysis reactor has a volume of 1 m³ ormore, preferably of more than 10 m³ and most preferably of 50 m³ ormore. In general the hydrolysis reactor will be smaller than 3000 m³ or5000 m³. The inventor poses the theory that especially at large scaleinsufficient oxygen is available for the hydrolysis which might be dueto oxygen transfer limitations in the reactor for example in thecellulolytic biomass. On lab-scale experiments this oxygen insufficiencymay play a less important role. The surface area (or oxygen contact areaof the reactor content) to reactor volume ratio is more favourable forsmall scale experiments than in large scale experiments. Moreover mixingin small scale experiments is relatively easier than at large scale.During those small scale experiments also the transport of oxygen fromthe headspace of the hydrolysis reactor is faster than compared to thesituation in large scale experiments. This theory is only given aspossible explanation of the effect noticed by the inventors, and thepresent invention does not fall or stands with the correctness of thistheory. According to a further embodiment of the invention the additionof oxygen may be used to control at least partially the hydrolysisprocess.

The process of the invention is advantageously applied in combinationwith the use of thermostable enzymes.

A “thermostable” enzyme means that the enzyme has a temperature optimum60° C. or higher, for example 70° C. or higher, such as 75° C. orhigher, for example 80° C. or higher such as 85° C. or higher. They mayfor example be isolated from thermophilic microorganisms, or may bedesigned by the skilled person and artificially synthesized. In oneembodiment the polynucleotides may be isolated or obtained fromthermophilic or thermotolerant filamentous fungi or isolated fromnon-thermophilic or non-thermotolerant fungi but are found to bethermostable.

By “thermophilic fungus” is meant a fungus that grows at a temperatureof 50° C. or above. By “themotolerant” fungus is meant a fungus thatgrows at a temperature of 45° C. or above, having a maximum near 50° C.

Examples of thermophilic fungal strains are Rasamsonia emersonii(formerly known as Talaromyces emersoni; Talaromyces emersonii,Penicillium geosmithia emersonii and Rasamsonia emersonii are usedinterchangeably herein).

Suitable thermophilic or thermotolerant fungal cells may be a Humicola,Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces,Thermoascus or Thielavia cell, preferably a Rasamsonia emersonii cell.Preferred thermophilic or thermotolerant fungi are Humicola grisea var.thermoidea, Humicola lanuginosa, Myceliophthora thermophila, Papulasporathermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii,Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata,Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei,Talaromyces bacillisporus, Talaromyces leycettanus, Talaromycesthermophilus, Thermomyces lenuginosus, Thermoascus crustaceus,Thermoascus thermophilus Thermoascus aurantiacus and Thielaviaterrestris.

Thermophilic fungi are not restricted to a specific taxonomic order andoccur all over the fungal tree of life. Examples are Rhizomucor in theMucorales, Myceliophthora in Sordariales and Talaromyces, Thermomycesand Thermoascus in the Eurotiales (Mouchacca 1997). The majority ofTalaromyces species are rnesophiles but exceptions are species withinsections Emersorii and Thermophila. Section Emersonii includesTalaromyces emersonii, Talaromyces byssochlamydoides, Talaromycesbacillisporus and Talaromyces leycettanus, all of which grow well at 40°C. Talaromyces bacillisporus is thermotolerant, T. leycettanus isthermotolerant to thermophilic, and T. emersonii and T.byssochlamydoides are truly thermophilic (Stolk and Samson 1972). Thesole member of Talaromyces section Thermophila, T. thermophilus, growsrapidly at 50° C. (Evans and Stolk 1971; Evans 1971; Stolk and Samson1972). The current classification of these thermophilic Talaromycesspecies is mainly based on phenotypic and physiological characters, suchas their ability to grow above 40° C., ascospore color, the structure ofascornatal covering and the formation of a certain type of anamorph.Stolk and Samson (1972) stated that the members of the section Emersoniihave anamorphs of either Paecilomyces (T. byssochlamydoides and T.leycettanus) or Penicillium cylindrosporum series (T. emersonii and T.bacillisporus). Later, Pitt (1979) transferred the species belonging tothe Penicillium cylindrosporum series to the genus Geosmithia, based onvarious characters such as the formation of conidia from terminal poresinstead of on collula (necks), a character of Penicillium andPaecilomyces. Within the genus Geosmithia, only G. argillacea isthermotolerant, and Stolk et al. (1969) and Evans (1971) proposed aconnection with members of Talaromyces sect. Emersonii. The phylogeneticrelationship of the themophilic Talaromyces species within Talaromycesand the Trichocomaceae is unknown. See J. Houbraken, Antonie vanLeeuwenhoek 2012 February; 101(2): 403-21.

Rasamsonia is a new genus comprising thermotolerant and thermophilicTalaromyces and Geosmithia species (J. Houbraken et al vida supra).Based on phenotypic, physiological and molecular data, Houbraken et alproposed to transfer the species T. emersonii, T. byssochlamydoides, T.eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. nov.Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsoniaemersonii are used interchangeably herein.

Preferred thermophilic fungi are Rasamsonia byssochlamydoides,Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus,Thermoascus crustaceus, Thermoascus thermophilus and Thermoascusaurantiacus.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium,Geosmithia, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Rasamsonia, Schizophyllum, Talaromyces,Thermoascus, Thermomyces, Thielavia, Tolypocladium, and Trichoderma.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.

An advantage of expression and production of the enzymes (for example atleast two, three or four different cellulases) in a suitablemicroorganism may be a high enzyme composition yield which can be usedin the process of the present invention.

According to the invention, by the addition of oxygen it is possible toattain many process advantages, including optimal temperatureconditions, reduced process time, reduced dosage of enzyme, re-use ofenzymes and other process optimizations, resulting in reduced costs.Advantageously the invention provides a process in which the hydrolysisstep is conducted at improved conditions. The invention also provides aprocess involving hydrolysis having a reduced process time. Furthermorethe invention provides a process, wherein the dosage of enzyme may bereduced and at the same time output of useful hydrolysis product ismaintained at the same level. Another advantage of the invention is thatthe present process involving hydrolysis may result in processconditions which are optimized. A still further advantage of theinvention is that the output of useful hydrolysis product of the processinvolving hydrolysis is increased using the same enzyme dosage.

Stable Enzyme Composition

Stable enzyme composition herein means that the enzyme compositionretains activity after 30 hours of hydrolysis reaction time, preferablyat least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%97%, 98%, 99% or 100% of its initial activity after 30 hours ofhydrolysis reaction time. Preferably the enzyme composition retainsactivity after 40, 50, 60, 70, 80, 90 100, 150, 200, 250, 300, 350, 400,450, 500 hours of hydrolysis reaction time.

The enzyme composition may be prepared by fermentation of a suitablesubstrate with a suitable microorganism, e.g. Rasamsonia emersonii orAspergillus niger wherein the enzyme composition is produced by themicroorganism. The microorganism may be altered to improve or to makethe cellulase composition. For example the microorganism may be mutatedby classical strain improvement procedures or by recombinant DNAtechniques. Therefore the microorganisms mentioned herein can be used assuch to produce the cellulase composition or may be altered to increasethe production or to produce an altered cellulase composition whichmight include heterologous cellulases, thus enzymes that are notoriginally produced by that microorganism. Preferably a fungus, morepreferably a filamentous fungus is used to produce the cellulasecomposition. Advantageously a thermophilic or thermotolerantmicroorganism is used. Optionally a substrate is used that induces theexpression of the enzymes in the enzyme composition during theproduction of the enzyme composition.

The enzyme composition is used to release sugars from lignocellulose,that comprises polysaccharides. The major polysaccharides are cellulose(glucans), hem icelluloses (xylans, heteroxylans and xyloglucans). Inaddition, some hemicellulose may be present as glucomannans, for examplein wood-derived feedstocks. The enzymatic hydrolysis of thesepolysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert. By sugar product is meant theenzymatic hydrolysis product of the feedstock or ligno-cellulosicmaterial. The sugar product will comprise soluble sugars, including bothmonomers and multimers, preferably will comprise glucose. Examples ofother sugars are cellobiose, xylose, arabinose, galactose, fructose,mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and otherhexoses and pentoses. The sugar product may be used as such or may befurther processed for example purified.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to a) generatesstructures more prone to interstrand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

Enzymes that may be included in the stable enzyme composition used inthe invention are now described in more detail:

GH61, Endoglucanases (EG) and exo-cellobiohydrolases (CBH) catalyze thehydrolysis of insoluble cellulose to products such ascellooligosaccharides (cellobiose as a main product), whileβ-glucosidases (BG) convert the oligosaccharides, mainly cellobiose andcellotriose to glucose.

Hemicellulose is a complex polymer, and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at 0 to 3and/or 0 to 2 atom of xylose, and can be substituted with linkages toarabinose, galactose, mannose, glucuronic acid, galacturonic acid or byesterification to acetic acid (and esterification of ferulic acid toarabinose). Hemicellulose can also contain glucan, which is a generalterm for β-linked six carbon sugars (such as the β-(1,3)(1,4) glucansand heteroglucans mentioned previously) and additionally glucomannans(in which both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis ofhemicelluloses.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of β(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another.

The principal types of structural unit are: galacturonan(homogalacturonan), which may be substituted with methanol on thecarboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI),in which galacturonic acid units alternate with rhamnose units carrying(1,4)-linked galactan and (1,5)-linked arabinan side-chains. Thearabinan side-chains may be attached directly to rhamnose or indirectlythrough the galactan chains; xylogalacturonan, with single xylosyl unitson O-3 of galacturonic acid (closely associated with RGI); andrhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

A composition for use in a method of the invention comprises preferablyat least two activities, although typically a composition will comprisemore than two activities, for example, three, four, five, six, seven,eight, nine or more. Typically, a composition of the invention maycomprise at least two different celulases or one cellulase and at leastone hemicellulase. A composition of the invention may comprisecellulases, but no xylanases. In addition, a composition of theinvention may comprise auxiliary enzyme activity, i.e. additionalactivity which, either directly or indirectly leads to lignocellulosedegradation. Examples of such auxiliary activities are mentioned herein.

Thus, a composition for use in the invention may comprise GH61,endoglucanase activity and/or cellobiohydrolase activity and/orβ-glucosidase activity. A composition for use in the invention maycomprise more than one enzyme activity in one or more of those classes.For example, a composition for use in the invention may comprise twoendoglucanase activities, for example, endo-1,3(1,4)-β glucanaseactivity and endo-β-1,4-glucanase activity. Such a composition may alsocomprise one or more xylanase activities. Such a composition maycomprise an auxiliary enzyme activity.

A composition for use in the invention may be derived from Rasamsoniaemersonii. In the invention, it is anticipated that a core set of(lignocellulose degrading) enzyme activities may be derived fromRasamsonia emersonii. Rasamsonia emersonii can provide a highlyeffective set of activities as demonstrated herein for the hydrolysis oflignocellulosic biomass. That activity can then be supplemented withadditional enzyme activities from other sources. Such additionalactivities may be derived from classical sources and/or produced by agenetically modified organism.

The activities in a composition for use in the invention may bethermostable. Herein, this means that the activity has a temperatureoptimum of about 60° C. or higher, for example about 70° C. or higher,such as about 75° C. or higher, for example about 80° C. or higher suchas 85° C. or higher. Activities in a composition for use in theinvention will typically not have the same temperature optima, butpreferably will, nevertheless, be thermostable.

In addition, enzyme activities in a composition for use in the inventionmay be able to work at low pH. For the purposes of this invention, lowpH indicates a pH of about 5.5 or lower, about 5 or lower, about 4.9 orlower, about 4.8 or lower, about 4.7 or lower, about 4,6 or lower, about4.5 or lower, about 4.4 or lower, about 4.3 or lower, about 4.2 orlower, about 4,1 or lower, about 4.0 or lower about 3.9 or lower, orabout 3.8 or lower, about 3.7 or lower, about 3.6 or lower, or about 3.5or lower.

Activities in a composition for use in the invention may be defined by acombination of any of the above temperature optima and pH values.

The composition used in a method of the invention may comprise, inaddition to the activities derived from Rasamsonia, a cellulase (forexample one derived from a source other than Rasamsonia) and/or ahemicellulase (for example one derived from a source other thanRasamsonia) and/or a pectinase.

A composition for use in the invention may comprise one, two, three,four classes or more of cellulase, for example one, two three or four orall of a GH61, an endoglucanase (EG), one or two exo-cellobiohydrolase(CBH) and a β-glucosidase (BG). A composition for use in the inventionmay comprise two or more of any of these classes of cellulase.

A composition of the invention may comprise an activity which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by the composition for usein a method of the invention. For example, a composition of theinvention may comprise one type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by a composition asdescribed herein and a second type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by an additionalcellulose/hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degrading ormodifying cellulose. A polypeptide which is capable of degradingcellulose is one which is capable of catalysing the process of breakingdown cellulose into smaller units, either partially, for example intocellodextrins, or completely into glucose monomers. A cellulaseaccording to the invention may give rise to a mixed population ofcellodextrins and glucose monomers when contacted with the cellulase.Such degradation will typically take place by way of a hydrolysisreaction.

GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV)proteins are oxygen-dependent polysaccharide monooxygenases (PMO's)according to the latest literature. Often in literature these proteinsare mentioned to enhance the action of cellulases on lignocellulosesubstrates. GH61 was originally classified as endogluconase based onmeasurement of very weak endo-1,4β-d-glucanase activity in one familymember. The term “GH61” as used herein, is to be understood as a familyof enzymes, which share common conserved sequence portions and foldingsto be classified in family of the well-established CAZY GHclassification system (http://www.cazy.org/GH61.html). The glycosidehydrolase family 61 is a member of the family of glycoside hydrolases EC3.2.1. GH61 is used herein as being part of the cellulases.

Herein, a hemicellulase is any polypeptide which is capable of degradingor modifying hemicellulose. That is to say, a hemicellulase may becapable of degrading or modifying one or more of xylan, glucuronoxylan,arabinoxylan, glucomannan and xyloglucan. A polypeptide which is capableof degrading a hemicellulose is one which is capable of catalysing theprocess of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase according to the invention may give rise to a mixedpopulation of oligosaccharides and sugar monomers when contacted withthe hemicellulase. Such degradation will typically take place by way ofa hydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading ormodifying pectin. A polypeptide which is capable of degrading pectin isone which is capable of catalysing the process of breaking down pectininto smaller units, either partially, for example into oligosaccharides,or completely into sugar monomers. A pectinase according to theinvention may give rise to a mixed population of oligosacchardies andsugar monomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

Accordingly, a composition of the invention may comprise any cellulase,for example, a GH61, a cellobiohydrolase, an endo-β1,4-glucanase, aβ-glucosidase or a β-(1,3)(1,4)-glucanase.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the ends of thechains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.

Herein, a β-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans glucans, but not on β-D-glucanscontaining only 1,3- or 1,4-bonds. This enzyme may also be referred toas licheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucanse when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, forexample, an endoxylanase, a β-xylosidase, a α-L-arabionofuranosidase, anα-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, acoumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanaseor a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endohydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

Herein, an β-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an β-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyse 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of catalyzing the deacetylation of xylans andxylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically,not from triacetylglycerol. Such a polypeptide typically does not act onacetylated mannan or pectin.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form: coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be, for example, anoligosaccharide or a polysaccharide. This enzyme may also be referred toas trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroylesterase or p-coumaric acid esterase. This enzyme also falls within EC3.1.1.73 so may also be referred to as a feruloyl esterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1→4)-β-D-galactanase or lactase.

Herein, a β-mannanase (EC 3.2.1.78) is any polypeptide which is capableof catalyzing the random hydrolysis of 1,4β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A composition of the invention may comprise any pectinase, for examplean endo polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha rhamnosidase, anexo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase,pectin polygalacturonase, poly-α-1,4-galacturonide glycanohydrolase,endogalacturonase; endo-D-galacturonase or poly(1,4-α-D-galacturonide)glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O=n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GaIUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be know asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

A composition of the invention will typically comprise at least onecellulase and/or at least one hemicellulase and/or at least onepectinase (one of which is a polypeptide according to the invention). Acomposition of the invention may comprise a GH61, a cellobiohydrolase,an endoglucanase and/or a β-glucosidase. Such a composition may alsocomprise one or more hemicellulases and/or one or more pectinases.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase or an expansin or a cellulose induced protein or acellulose integrating protein or like protein may be present in acomposition of the invention (these are referred to as auxiliaryactivities above).

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases. “Lipase” includes enzymes that hydrolyze lipids,fatty acids, and acylglycerides, including phospoglycerides,lipoproteins, diacylglycerols, and the like. In plants, lipids are usedas structural components to limit water loss and pathogen infection.These lipids include waxes derived from fatty acids, as well as cutinand suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalyzing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

A composition for use in the invention may comprise an expansin orexpansin-like protein, such as a swollenin (see Salheimo et al., Eur. J.Biohem. 269, 4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition for use in the invention may be a cellulose inducedprotein, for example the polypeptide product of the cip1 or cip2 gene orsimilar genes (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997,2003), a cellulose/cellulosome integrating protein, for example thepolypeptide product of the cipA or cipC gene, or a scaffoldin or ascaffoldin-like protein. Scaffoldins and cellulose integrating proteinsare multi-functional integrating subunits which may organizecellulolytic subunits into a multi-enzyme complex. This is accomplishedby the interaction of two complementary classes of domain, i.e. acohesion domain on scaffoldin and a dockerin domain on each enzymaticunit. The scaffoldin subunit also bears a cellulose-binding module (CBM)that mediates attachment of the cellulosome to its substrate. Ascaffoldin or cellulose integrating protein for the purposes of thisinvention may comprise one or both of such domains.

A composition for use in a method of the invention may be composed of amember of each of the classes of enzymes mentioned above, severalmembers of one enzyme class, or any combination of these enzymes classesor helper proteins (i.e. those proteins mentioned herein which do nothave enzymatic activity per se, but do nevertheless assist inlignocellulosic degradation).

A composition for use in a method of the invention may be composed ofenzymes from (1) commercial suppliers; (2) cloned genes expressingenzymes; (3) complex broth (such as that resulting from growth of amicrobial strain in media, wherein the strains secrete proteins andenzymes into the media; (4) cell lysates of strains grown as in (3);and/or (5) plant material expressing enzymes. Different enzymes in acomposition of the invention may be obtained from different sources.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to lignocellulosic feedstock. Alternatively, the enzymes are produced,but not isolated, and crude cell mass fermentation broth, or plantmaterial (such as corn stover or wheat straw), and the like may be addedto, for example, the feedstock. Alternatively, the crude cell mass orenzyme production medium or plant material may be treated to preventfurther microbial growth (for example, by heating or addition ofantimicrobial agents), then added to, for example, a feedstock. Thesecrude enzyme mixtures may include the organism producing the enzyme.Alternatively, the enzyme may be produced in a fermentation that uses(pre-treated) feedstock (such as corn stover or wheat straw) to providenutrition to an organism that produces an enzyme(s). In this manner,plants that produce the enzymes may themselves serve as alignocellulosic feedstock and be added into lignocellulosic feedstock.

In the uses and methods described herein, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The invention thus relates to methods in which the composition describedabove are used and to uses of the composition in industrial processes.

Ligno-Cellulosic Material

Lignocellulosic material herein includes any lignocellulosic and/orhemicellulosic material. Lignocellulosic material suitable for use asfeedstock in the invention includes biomass, e.g. virgin biomass and/ornon-virgin biomass such as agricultural biomass, commercial organics,construction and demolition debris, municipal solid waste, waste paperand yard waste. Common forms of biomass include trees, shrubs andgrasses, wheat, wheat straw, sugar cane bagasse, switch grass,miscanthus, corn, corn stover, corn husks, corn cobs, canola stems,soybean stems, sweet sorghum, corn kernel including fiber from kernels,products and by-products from milling of grains such as corn, wheat andbarley (including wet milling and dry milling) often called “bran orfibre” as well as municipal solid waste, waste paper and yard waste. Thebiomass can also be, but is not limited to, herbaceous material,agricultural residues, forestry residues, municipal solid wastes, wastepaper, and pulp and paper mill residues. “Agricultural biomass” includesbranches, bushes, canes, corn and corn husks, energy crops, forests,fruits, flowers, grains, grasses, herbaceous crops, leaves, bark,needles, logs, roots, saplings, short rotation woody crops, shrubs,switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp,wheat midlings, oat hulls, and hard and soft woods (not including woodswith deleterious materials). In addition, agricultural biomass includesorganic waste materials generated from agricultural processes includingfarming and forestry activities, specifically including forestry woodwaste. Agricultural biomass may be any of the aforementioned singularlyor in any combination or mixture thereof.

Pre-Treatment

The feedstock may optionally be pre-treated with heat, mechanical and/orchemical modification or any combination of such methods in order to toenhance the accessibility of the substrate to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in any way known in the art. In oneembodiment, the pre-treatment is conducted treating the lignocellulosewith steam explosion, hot water treatment or treatment with dilute acidor dilute base.

Washing Step

Optionally, the process according to the invention comprises a washingstep. The optional washing step may be used to remove water solublecompounds that may act as inhibitors for the fermentation step. Thewashing step may be conducted in known manner.

Enzymatic Hydrolysis

The enzyme composition used in the process of the invention canextremely effectively hydrolyze lignocellulolytic material, for examplecorn stover or wheat straw, which can then be further converted into auseful product, such as ethanol, biogas, butanol, lactic acid, aplastic, an organic acid, a solvent, an animal feed supplement, apharmaceutical, a vitamin, an amino acid, an enzyme or a chemicalfeedstock. Additionally, intermediate products from a process followingthe hydrolysis, for example lactic acid as intermediate in biogasproduction, can be used as building block for other materials. Thepresent invention is exemplified with the production of ethanol but thisis done as exemplification only rather than as limitation, the othermentioned useful products can be produced equally well.

The process according to the invention comprises an enzymatic hydrolysisstep. The enzymatic hydrolysis includes, but is not limited to,hydrolysis for the purpose of liquification of the feedstock andhydrolysis for the purpose of releasing sugar from the feedstock orboth. In this step optionally pre-treated and optionally washedligno-cellulosic material is brought into contact with the enzymecomposition according to the invention. Depending on the lignocellulosicmaterial and the pre-treatment, the different reaction conditions, e.g.temperature, enzyme dosage, hydrolysis reaction time and dry matterconcentration, may be adapted by the skilled person in order to achievea desired conversion of lignocellulose to sugar. Some indications aregiven hereafter.

In one aspect of the invention the hydrolysis is conducted at atemperature of 45° C. or more, 50° C. or more, 55° C. or more, 60° C. ormore, 65° C. or more, or 70° C. or more. The high temperature duringhydrolysis has many advantages, which include working at the optimumtemperature of the enzyme composition, the reduction of risk of(bacterial) contamination, reduced viscosity, smaller amount of coolingwater required, use of cooling water with a higher temperature, re-useof the enzymes and more.

In a further aspect of the invention, the amount of enzyme compositionadded (herein also called enzyme dosage or enzyme load) is low. In anembodiment the amount of enzyme is 6 mg protein/g dry matter weight orlower, 5 mg protein/g dry matter or lower, 4 mg protein/g dry matter orlower, 3 mg protein/g dry matter or lower, 2 mg protein/g dry matter orlower, or 1 mg protein/g dry matter or lower (expressed as protein in mgprotein/g dry matter). In an embodiment, the amount of enzyme is 0.5 mgenzyme/g dry matter weight or lower, 0.4 mg enzyme composition/g drymatter weight or lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25mg enzyme/g dry matter weight or lower, 0.20 mg enzyme/g dry matterweight or lower, 0.18 mg enzyme /g dry matter weight or lower, 0.15 mgenzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matterweight or lower (expressed as total of cellulase enzymes in mg enzyme/gdry matter). Low enzyme dosage is possible, since because of theactivity and stability of the enzymes, it is possible to increase thehydrolysis reaction time.

In a further aspect of the invention, the hydrolysis reaction time is 5hours or more, 10 hours or more, 20 hours or more, 40 hours or more, 50hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90hours or more, 100 hours or more, 120 hours or more, 130 h or more. Inanother aspect, the hydrolysis reaction time is 5 to 150 hours, 40 to130 hours, 50 to 120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100hours, 70 to 100 hours, 70 to 90 hours or 70 to 80 hours. Due to thestability of the enzyme composition longer hydrolysis reaction times arepossible with corresponding higher sugar yields.

The pH during hydrolysis may be chosen by the skilled person. In afurther aspect of the invention, the pH during the hydrolysis may be 3.0to 6.4. The stable enzymes of the invention may have a broad pH range ofup to 2 pH units, up to 3 pH units, up to 5 pH units. The optimum pH maylie within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to6.0, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.

In a further aspect of the invention the hydrolysis step is conducteduntil 70% or more, 80% or more, 85% or more, 90% or more, 92% or more,95% or more of available sugar in lignocellulosic material is released.

Significantly, a process of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. Thus, the invention may be carried out with a dry mattercontent of about 5 wt % or higher, about 8 wt % or higher, about 10 wt %or higher, about 11 wt % or higher, about 12 wt % or higher, about 13 wt% or higher, about 14 wt % or higher, about 15 wt % or higher, about 20wt % or higher, about 25 wt % or higher, about 30 wt % or higher, about35 wt % or higher or about 40 wt % or higher. In a further embodiment,the dry matter content in the hydrolysis step is 14 wt %, 15 wt %, 16 wt%, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt%, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt%, 33 wt % or more or 14 to 33 wt %.

Fermentation

The process according to the invention comprises a fermentation step. Ina further aspect, the invention thus includes in step fermentationprocesses in which a microorganism is used for the fermentation of acarbon source comprising sugar(s), e.g. glucose, L-arabinose and/orxylose. The carbon source may include any carbohydrate oligo- or polymercomprising L-arabinose, xylose or glucose units, such as e.g.lignocellulose, xylans, cellulose, starch, arabinan and the like. Forrelease of xylose or glucose units from such carbohydrates, appropriatecarbohydrases (such as xylanases, glucanases, amylases and the like) maybe added to the fermentation medium or may be produced by the modifiedhost cell. In the latter case the modified host cell may be geneticallyengineered to produce and excrete such carbohydrases. An additionaladvantage of using oligo- or polymeric sources of glucose is that itenables to maintain a low(er) concentration of free glucose during thefermentation, e.g. by using rate-limiting amounts of the carbohydrases.This, in turn, will prevent repression of systems required formetabolism and transport of non-glucose sugars such as xylose. In apreferred process the modified host cell ferments both the L-arabinose(optionally xylose) and glucose, preferably simultaneously in which casepreferably a modified host cell is used which is insensitive to glucoserepression to prevent diauxic growth. In addition to a source ofL-arabinose, optionally xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the modified host cell. Compositions offermentation media for growth of microorganisms such as yeasts orfilamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50g/l glucose and corresponding other sugars from the lignocellulosicfeedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.For more dilute sugar compositions the fermentation time maycorrespondingly be reduced.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD⁺. Thus, in a preferred anaerobic fermentationprocess pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation process is anaerobic. Ananaerobic process is advantageous since it is cheaper than aerobicprocesses: less special equipment is needed. Furthermore, anaerobicprocesses are expected to give a higher product yield than aerobicprocesses. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation process is under oxygen-limitedconditions. More preferably, the fermentation process is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation processis a process in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a process under oxygen-limitedconditions, the rate of oxygen consumption is at least 5.5, morepreferably at least 6 and even more preferably at least 7 mmol/L/h.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably less than 38° C. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C.

In an embodiment of the invention, in step the fermentation is conductedwith a microorganism that is able to ferment at least one C5 sugar. Inan embodiment the process is a process for the production of ethanolwhereby the process comprises the step comprises fermenting a mediumcontaining sugar(s) with a microorganism that is able to ferment atleast one C5 sugar, whereby the host cell is able to ferment glucose,L-arabinose and xylose to ethanol. In an embodiment thereof themicroorganism that is able to ferment at least one C5 sugar is a yeast.In an embodiment, the yeast is belongs to the genus Saccharomyces,preferably of the species Saccharomyces cerevisiae, in which geneticmodifications have been made. An example of such a microorganism and itspreparation is described in more detail in WO 2008/041840 and inEuropean Patent Application EP10160622.6, filed 21 Apr. 2010. In anembodiment, the fermentation process for the production of ethanol isanaerobic. Anaerobic has already been defined earlier herein. In anotherpreferred embodiment, the fermentation process for the production ofethanol is aerobic. In another preferred embodiment, the fermentationprocess for the production of ethanol is under oxygen-limitedconditions, more preferably aerobic and under oxygen-limited conditions.Oxygen-limited conditions have already been defined earlier herein.

In such process, the volumetric ethanol productivity is preferably atleast 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre perhour. The ethanol yield on L-arabinose and optionally xylose and/orglucose in the process preferably is at least 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as apercentage of the theoretical maximum yield, which, for glucose andL-arabinose and optionally xylose is 0.51 g. ethanol per g. glucose orxylose.

In one aspect, the fermentation process leading to the production ofethanol, has several advantages by comparison to known ethanolfermentations processes:

anaerobic processes are possible;

oxygen limited conditions are also possible;

higher ethanol yields and ethanol production rates can be obtained;

the strain used may be able to use L-arabinose and optionally xylose.

Alternatively to the fermentation processes described above, at leasttwo distinct cells may be used, this means this process is aco-fermentation process. All preferred embodiments of the fermentationprocesses as described above are also preferred embodiments of thisco-fermentation process: identity of the fermentation product, identityof source of L-arabinose and source of xylose, conditions offermentation (aerobical or anaerobical conditions, oxygen-limitedconditions, temperature at which the process is being carried out,productivity of ethanol, yield of ethanol).

The fermentation process may be carried out without any requirement toadjust the pH during the process. That is to say, the process is onewhich may be carried out without the addition of any acid(s) or base(s).However, this excludes a pretreatment step, where acid may be added. Thepoint is that the composition of the invention is capable of acting atlow pH and, therefore, there is no need to adjust the pH of acid of anacid pretreated feedstock in order that saccharification or hydrolysismay take place. Accordingly, a method of the invention may be a zerowaste method using only organic products with no requirement forinorganic chemical input.

Overall Reaction Time

According to the invention, the overall reaction time (or the reactiontime of hydrolysis step and fermentation step together) may be reduced.In one embodiment, the overall reaction time is 300 hours or less, 200hours or less, 150 hours or less, 140 hours or less, 130 or less, 120hours or less, 110 hours or less, 100 hours of less, 90 hours or less,80 hours or less, 75 hours or less, or about 72 hours at 90% glucoseyield. Correspondingly lower overall times may be reached at lowerglucose yield.

Fermentation Products

Fermentation products which may be produced according to the inventioninclude amino acids, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels, orother organic polymers, lactic acid, and ethanol, including fuel ethanol(the term “ethanol” being understood to include ethyl alcohol ormixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of theinvention include, but not limited to, biofuels (including biogas,ethanol and butanol); lactic acid; 3-hydroxy-propionic acid; acrylicacid; acetic acid; 1,3-propane-diol; ethylene; glycerol; a plastic; aspecialty chemical; an organic acid, including citric acid, succinicacid and maleic acid; a solvent; an animal feed supplement; apharmaceutical such as a β-lactam antibiotic or a cephalosporin; avitamin; an amino acid, such as lysine, methionine, tryptophan,threonine, and aspartic acid; an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase; a chemical feedstock; or ananimal feed supplement.

Separation of Fermentation Product

The process according to the invention optionally comprises recovery offermentation product. A fermentation product may be separated from thefermentation broth in any known manner. For each fermentation productthe skilled person will thus be able to select a proper separationtechnique. For instance ethanol may be separated from a yeastfermentation broth by distillation, for instance steamdistillation/vacuum distillation in conventional way.

Certain embodiments of the invention will below be described in moredetail, but are in no way limiting the scope of the present invention.

Use of Thermostable Enzymes under Optimal Temperature Conditions

In one embodiment, the invention relates to the use of thermostableenzymes such as cellulolytic enzymes of Rasamsonia for the production ofreducing sugars from pre-treated ligno-cellulosic feedstock in, but notlimiting to, ethanol production. Cellulolytic enzymes of Rasamsoniaapplied on pre-treated ligno-cellulosic feedstock showed maximalconversion rates at temperature within the range of 50 to 70° C. Theenzyme remains active under these circumstances for 14 days and morewithout complete cessation of activity.

By using optimal temperature conditions, maximal amount of reducingsugars can be released from feedstock (total hydrolysis) within theshortest possible hydrolysis time. In this way, 100% conversion ofcellulose in glucose is achieved in less than 5 days.

The theoretical maximum yield (Yps max in g product per gram glucose) ofa fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 g) yields according to normal glycolysisfermentation pathway in yeast 2 moles of ethanol (=2×46=92 g ethanol.The theoretical maximum yield of ethanol on glucose is therefore92/180=0.511 g ethanol/g glucose.

For butanol (MW 74 g/mole) or iso butanol, the theoretical maximum yieldis 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 g (iso-)butanol/g glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 g/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 g lactic acid/g glucose.

For other fermentation products a similar calculation may be made.

The cost reduction achieved with applying cellulolytic enzymes ofRasamsonia will be the result of an overall process time reduction.

Compensation of Lower Enzyme Dosage with Extended Hydrolysis Time UsingRasamsonia Enzymes

Due to the high stability of the stable enzymes, the activities do notcease in time, although less reducing sugars are liberated in the courseof the hydrolysis. It is possible to lower the enzyme dosage and extendthe use of the enzyme by prolonging the hydrolysis times to obtainsimilar levels of released reducing sugars. For example, 0.175 mLenzyme/g feedstock dry-matter resulted in release of approximately 90%of the theoretical maximum of reducing sugars from pre-treated feedstockwithin 72 h. When using 0.075 mL enzyme/g feedstock dry-matter,approximately 90% conversion of the theoretical maximum is achievedwithin 120 h. The results show that, because of the stability of theenzyme activity, lowering the enzyme dosage can be compensated byextending the hydrolysis time to obtain the same amount of reducingsugars. The same holds for hydrolysis of pre-treated feedstock atdry-matter contents higher than 10% shows that compensating effect ofextended hydrolysis time at 15% dry matter feedstock.

The cost reduction achieved by using stable cellulolytic enzymes, suchas of Rasamsonia, results from requiring less enzyme dosage, resultingin similar hydrolysis conversion yields.

Lowering the Risk on Contamination with Stable Enzymes

In a common process for converting ligno-cellulosic material intoethanol, process steps are preferably done under septic conditions tolower the operational costs. Contamination and growth of contaminatingmicroorganisms can therefore occur and result in undesirable sideeffects, such lactic acid, formic acid and acetic acid production, yieldlosses of ethanol on substrate, production of toxins and extracellularpolysaccharides, which may affect production costs significantly. A highprocess temperature and/or a short process time will limit the risk oncontamination during hydrolysis and fermentation. Thermostable enzymes,like those of Rasamsonia, are capable of hydrolysing ligno-cellulosicfeedstock at temperatures of higher than 60° C. At these temperatures,the risk that a contaminating microorganism will cause undesired sideeffects will be little to almost zero.

During the fermentation step, in which ethanol is produced, temperaturesare typically between 30 to 37° C. and will preferably not be raisedbecause of production losses. By applying fermentation process times asshort as possible the risks and effects of contamination and/or growthof contaminants will be reduced as much as possible. With stableenzymes, like those of Rasamsonia a short as possible fermentation timescan be applied (see description above), and thus risks on contaminationand/or growth of contaminants will be reduced as much as possible. Thecost reduction achieved with applying thermostable cellulolytic enzymesof Rasamsonia in this way will result from lower risk of processfailures due to contamination.

Stable Enzymes Reduce Cooling Costs and Increase Productivity of EthanolPlants

The first step after thermal pretreatment will be to cool the pretreatedfeedstock to temperatures where the enzymes are optimal active. On largescale, this is typically done by adding (cooled) water, which will,besides decreasing the temperature, reduce the dry-matter content. Byusing thermos stable enzymes, like those of Rasamsonia, cost reductioncan be achieved by the fact that (i) less cooling of the pretreatedfeedstock is required since higher temperatures are allowed duringhydrolysis, and (ii) less water will be added, which will increase thedry-matter content during hydrolysis and fermentation and thus increasethe ethanol production capacity (amount produced per time unit pervolume) of an ethanol plant. Also, by using thermostable enzymesaccording to the invention, like those of Rasamsonia, cost reduction mayalso be achieved by using cooling water having higher temperature thatthe water that is used in a process with non-thermostable enzyme.

Enzyme Recycling after Hydrolysis with Stable Enzymes

At the end of the hydrolysis, enzyme activities appear to be low sincelittle reducing sugars are released once almost all cellulose isconverted. The amount of enzymatic activity present, however, hasdecreased only a little, assumingly mainly due to absorption of theenzymes to the substrate. By applying solid-liquid separation afterhydrolysis, such as centrifugation, filtration, sedicantation, etcetera,60% or more e.g. 70% of the enzyme activity in solution can be recoveredand re-used for hydrolysis of a new pre-treated ligno-cellulosicfeedstock during the next hydrolysis.

Moreover, after solid-liquid separation the enzyme in solution can beseparated from the solution containing reducing sugars and otherhydrolysis products from the enzymatic actions. This separation can bedone by, but not limiting to, (ultra and micro)filtration,centrifugation, sedicantation, sedimentation, with or without firstadsorption of the enzyme to a carrier of any kind.

For example, after hydrolysis of pre-treated feedstock with 0.175 mL/gfeedstock dry matter enzyme load for 20 h, 50% of the theoreticalmaximum amount of reducing sugars is liberated and after the samehydrolysis for 72 h, 90% of the theoretical maximum amount of reducingsugars is liberated. By centrifugation and ultrafiltration, 60-70% ofthe enzyme activity was recovered in the retentate, while the filtratecontained more than 80% of the liberated reducing sugars. By re-usingthe retentate, either as it is or after further purification and/orconcentration, enzyme dosage during the next hydrolysis step can bereduced with 60 to 70%. The cost reduction achieved by using stablecellulolytic enzymes, such as of Rasamsonia, in this way results fromrequiring less enzyme dosage.

Enzyme Recyclina after Hydrolysis in Combination with Enzyme Productionand Yeast-Cell Recyclina with Stable Enzymes

The process including enzyme recycling after hydrolysis, as describedabove, can be combined with recycling of the ethanol producingmicroorganism after fermentation and with the use of the reducing sugarscontaining filtrate as a substrate (purified and/or concentrated ordiluted) in enzyme-production fermentation and as substrate for thecultivation of the ethanol-producing microorganism.

Enzyme Recyclina after Vacuum Distillation with Stable Enzymes

The thermo stability of enzymes, like those from Rasamsonia, causesremaining cellulolytic activity after hydrolysis, fermentation andvacuum distillation in the thin stillage. The total activity of theenzyme is reduced during the three successive process steps. The thinstillage obtained after vacuum distillation can thus be re-used as asource of enzyme for a newly startedhydrolysis-fermentation-distillation process cycle of pre-treated wheatstraw conversion into ethanol. The thin stillage can be used either inconcentrated or (un)diluted form and/or purified and with or withoutadditional enzyme supplementation.

Enzyme Recycling in Combination with Enzyme Supplementation after VacuumDistillation with Thermostable Enzymes

In an optimal process, an amount of enzyme is supplemented into the thinstillage, before its re-use in a new process cycle, equal to the amountof activity lost during the three successive process steps of theprevious process cycle. In this way over-dosage of enzyme is avoided andthus most efficient use of enzyme is obtained.

Moreover, by providing high enzyme dosage in the first process cycle,and supplementing enzyme equal to the amount of activity lost during thethree successive process steps in the following process cycles, highestpossible hydrolysis rates can be obtained in each process cycleresulting in short hydrolysis times of less than 48 h in combinationwith most efficient use of enzymes.

Use of Stable Enzymes in Mixed Systems

By applying mixing during hydrolysis, enzymes come more often in contactwith substrates, which results in a more efficient use of the catalyticactivity. This will result in a lower enzyme dosages and thus in lowercosts, unless the mixing has a negative effect on the enzymes. Stableenzymes, like the thermostable enzymes from Rasamsonia, are robust andcan resist circumstances of (locally) high shear and temperatures, whichis the case during intensive mixing of slurries. The use of it in mixedsystems is therefore beneficial and will lead to dosage and thus costsreduction.

The invention is further described by the following examples, whichshould not be construed as limiting the scope of the invention.

EXAMPLES Experimental Information Strains

Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAALBUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 ADUtrecht, The Netherlands in December 1964 having the Accession NumberCBS 393.64. Other suitable strains can be equally used in the presentexamples to show the effect and advantages of the invention. For exampleTEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsoniastrains which are described in WO2011/000949.

Preparation of Acid Pre-Treated Corn Stover Substrate.

Dilute-acid pre-treated corn stover (aCS) was obtained as described inSchell, D. J., Applied Biochemistry and Biotechnology (2003), vol.105-108, pp 69-85. A pilot scale pretreatment reactor was used operatingat steady state conditions of 190° C., 1 min residence time and aneffective H2SO4 acid concentration of 1.45% (w/w) in the liquid phase.

Protein Measurement Assays 1.Total Protein TCA Biuret

The method was a combination of precipitation of protein using trichloroacetic acid (TCA) to remove disturbing substances and allowdetermination of the protein concentration with the colorimetric Biuretreaction. In the Biuret reaction, a copper (II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptidebonds in an alkaline solution. A violet color indicates the presence ofproteins. The intensity of the color, and hence the absorption at 546nm, is directly proportional to the protein concentration, according tothe Beer-Lambert law. The standardisation was performed using BSA(Bovine Serum Albumine) and the protein content was expressed in gprotein as BSA equivalent/L or mg protein as BSA equivalent /ml. Theprotein content was calculated using standard calculation protocolsknown in the art, by plotting the OD₅₄₆ versus the concentration ofsamples with known concentration, followed by the calculation of theconcentration of the unknown samples using the equation generated fromthe calibration line.

2. Individual Proteins Using PAGE Sample Pre-Treatment SDS-PAGE

Based on the estimated protein concentration of the samples thefollowing samples preparation was performed. To 10 μl sample 40 μlMilliQ water and 50 μl TCA (20%) was added to dilute the sample fivetimes (˜1 mg/ml) and precipitate the proteins. After 1 hour on ice thesample was centrifuged (10 minutes, 14000 rpm). The pellet was washedwith 500 μl Aceton and centrifuged (10 minutes, 14000 rpm). The pelletwas treated as described below.

SDS-PAGE

The pellet was dissolved in 65 μl of the MilliQ water, 25 μl NuPAGE™ LDSsample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing agent(10×) Invitrogen. Prior to the the deanuarion step the sample wasdiluted 5 timnes using a mix of MilliQ; NuPAGE™ LDS sample buffer and 10μl NuPAGE™ Sample Reducing in the ratio of 65:25:10. After mixing, thesamples were incubated in a thermo mixer for 10 minutes at 70° C. Thesample solutions were applied on a 4-12% Bis-Tris gel (NuPAGE™ BisTris,Invitrogen). A sample (10μl ) of marker M12 (Invitrogen) was alsoapplied on the gel. The gel was run at 200 V for 50 minutes, using theXCELL Surelock, with 600 ml 20 ×diluted SDS buffer in the outer bufferchamber and 200 ml 20× diluted SDS buffer, containing 0.5 ml ofantioxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. Afterrunning, the gel was rinsed twice with demineralised water the gels werefixed with 50% methanol/7% acetic acid solution for one hour and stainedwith Sypro Ruby (50 ml per gel) overnight. An image was made using theTyphoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) afterwashing the gel with MilliQ water.

Quantitative Analysis of the Protein

Using the Typhoon scanner the ratio between protein bands within a lanewas determined using standard methods known in the art. The sample wasapplied in triplicate and the gray values were determined using theprogram Image quant. Values are expressed as relative % protein to thetotal protein, calculated using the gray value of the selected proteinband relative to the total gray value all the protein bands.

Glucan Conversion Calculation:

glucan conversion(%)=(glucose(g/l)×100%)/(glucan(fraction onDM)×dm(g/kg)×1.1)

-   Wherein:-   glucose (g/l)=glucose concentration in supernatant after hydrolysis.-   glucan (fraction on dm)=glucan content of the substrate before    pretreatment.-   dm (g/kg)=dry matter of hydrolysis (f.i. 20% dm=200 g/kg).-   1.1=weight increase due to water incorporation during hydrolysis.

Example Calculation:

-   glucose=60 g/l-   glucan fraction=0.40 (is 40% on dry matter)-   dm=200 g/kg

glucan conversion example=(60*100)/(0.4×200×1.1)=68% conversion

Example 1 Evaluation of the Effect of the Absence of Oxygen DuringHydrolysis on the Cellulolytic Activity of Cellulase Enzyme Cocktails

The effect of oxygen absence during hydrolysis on the cellulolyticactivity of three different enzyme cocktails was evaluated according tothe procedures described below. The hydrolysis reactions were performedwith acid pretreated cornstover (aCS) feedstock at a final concentrationof 10 w/w % DM. This feedstock solution was prepared via the dilution ofa concentrated feedstock solution with water. Subsequently the pH wasadjusted to pH 4.5 with a 4M NaOH solution. The elimination of oxygenfrom the feedstock was accomplished in two steps. First, the feedstocksolution was degassed via sonication under vacuum in a sonication bath(Bransonic 5510E-DTH, setting; Degas) for 15 minutes. In the secondstep, the oxygen was further removed by continuous sparging of anitrogen flow through a 500 ml solution of the 10% DM feedstock for aperiod of 3 hours. Prior to being sparged through the feedstocksolution, the nitrogen flow was sparged through water in order tosaturate it with water vapour and prevent evaporation of the water fromthe feedstock solution. In parallel, 500 ml of the same batch 10 w/w %DM aCS was sparged with air as an oxygen-containing control sample in asimilar set-up and according to the same protocol.

The hydrolysis of the oxygen-depleted (nitrogen sparged) and theoxygen-saturated (air-sparged) 10 w/w % aCS feedstock solutions wereconducted in air-tight, 30-ml centrifuge bottles (Nalgene Oakridge) in atotal reaction volume of 10 ml. The bottles, already containing thecellulase solution, used for the oxygen-depleted experiment were spargedwith nitrogen prior to- and during filling them with feedstock. Eachhydrolysis was performed in duplicate with 7.5 mg/g DM cellulase enzymecocktail added in a total volume not larger than 375 μl. The threecellulase enzyme cocktails tested included: a TEC-210 mix (mixture ofcellulases), a 4E-GH61 mix (consisting of 9 w/w % of total protein BG,30 w/w % of total protein CBHI, 25 w/w % of total protein GBHII and 36w/w % of total protein GH61) and a 4E-EG mix (consisting of 9 w/w % oftotal protein BG, 30 w/w % of total protein CBHI, 25 w/w % of totalprotein CBHII and 36 w/w % of total protein EG). TEC-210 was fermentedaccording to the inoculation and fermentation procedures described inWO2011/000949. The 4E mix (as described in WO2011/098577) was used.

The centrifuge bottles containing the feedstock and enzyme solution wereplaced in an oven incubator (Techne HB-1 D hybridization oven) andincubated for 72 hours at 65° C. while rotating at set-point 3 (12 rpmper minute). Following hydrolysis, the samples were cooled on ice andimmediately 50 μl of each supernatant was diluted in 1450 μl grade Iwater. The diluted supernatant was subsequently filtered (0.45 μmfilter, Pall PN 454) and the filtrates were analysed for sugar contentas described below.

The sugar concentrations of the diluted samples were measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

The data presented in Table 1/FIG. 1 show that the glucose released fromthe nitrogen-sparged feedstocks is lower than the glucose released fromthe feedstocks sparged with air for both the TEC-210 mix and the 4E-GH61mix incubations. There is no difference in glucose release detectablebetween the nitrogen and air sparged feedstocks for samples hydrolyzedby the 4E-EG mix.

Based on these results we conclude that the presence of oxygen improvesthe cellulolytic performance of cellulase mixtures that contain GH61enzymes.

TABLE 1 The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose released bythree different cellulase mixes. Sparged with air Average glucoseSparged with N₂ Cellulase (g/l) stdev Average glucose (g/l) stdevTEC-210 34.5 0.8 31.9 1.1 4E-GH61 mix 31.7 1.4 27.4 0.1 4E-EG mix 22.70.1 23.3 1.7

Example 2 The Effect of the Dissolved Oxygen Concentration on theCellulolytic Activity of Cellulase Enzyme Compositions During Hydrolysisof Lignocellulosic Feedstock on Pilot Scale (270 liter)

The effect of the dissolved oxygen concentration on the cellulolyticactivity of the enzyme composition or enzyme cocktail during thehydrolysis of lignocellulosic feedstock on pilot scale (270 liter) isshown in this example. The hydrolysis reactions were performed with acidpretreated cornstover (aCS) feedstock at a (final) concentration of 20w/w % DM. The feedstock solution was prepared by the dilution ofconcentrated feedstock slurry with water. The pH was adjusted to pH 4.5with a 25% (w/w) NH₄OH solution.

The enzymatic hydrolysis was done in a 270 liter pilot reactor which waspH and temperature controlled with a working volume of 150 liter. Thedissolved oxygen during the process was controlled by adjusting impellerspeed at a given airflow and overpressure. The enzymatic hydrolysis wasperformed with a dosage of 3.75 mg TCA protein/g DM of TEC-210 cellulaseenzyme composition. TEC-210 was produced according to the inoculationand fermentation procedures described in WO2011/000949.

The following experiments were done:

-   1. 150 l of 20% aCS, pH 4.5, temperature 62° C., no overpressure, no    airflow, 3.75 mg TCA/g dm of TEC-210 cellulase composition,    incubation time 120 hours in a 270 liter pilot reactor. The    dissolved oxygen concentration (DO) of the reaction mixture was    measured constantly using a DO electrode. The DO was controlled at a    level of 0.01 mol/m3 by adjusting the impeller speed.-   2. 150 l of 20% aCS, pH 4.5, temperature 62° C., 1 bar overpressure,    10 kg/h airflow in the headspace of the reactor, 3.75 mg TCA/g dm of    TEC-210 cellulase composition, incubation time 120 hours in a 270    liter pilot reactor The dissolved oxygen concentration (DO) of the    reaction mixture was measured constantly using a DO electrode. The    DO was controlled at a level of 0.2 mol/m3 by adjusting the impeller    speed.

During the enzymatic hydrolysis, samples were taken daily forcarbohydrate analysis (glucose, cellobiose) by NMR and viscosity and pHmeasurement.

Composition analysis of the acid pretreated Corn Stover (aCS) was doneby chemical hydrolysis of the sample and determination of the monosaccharides by NMR.

Samples taken during enzymatic hydrolysis were analysed for(oligo)sugars, organic acids and inhibitors by flow NMR.

Conversion was calculated based on the measured glucan concentration(g/kg) at the start of the enzymatic hydrolysis and glucoseconcentration (g/l) which was measured during the enzymatic hydrolysis.

The results are presented in FIG. 2.

This example shows that at larger reactor volumes, for example 100 literor more the addition of oxygen (in the form of air) strongly improvesthe hydrolysis of pretreated lignocellulosic material. On scales of 1 m³or more, or 10 m³ or more or 50 m³ or more reactor content similarimprovement in conversion during hydrolysis will be found.

Example 3 The Effect of Oxygen on the Cellulolytic Activity of CellulaseEnzyme Compositions During Hydrolysis of Lignocellulosic Feedstock usinga Low Enzyme Dosage

The effect of oxygen on the cellulolytic activity of the enzymecomposition using a low enzyme dosage during the hydrolysis oflignocellulosic feedstock is shown in this example. The hydrolysisreactions are performed with acid pretreated cornstover (aCS) feedstockat a final concentration of 20 w/w % DM. This feedstock solution isprepared via the dilution of a concentrated feedstock solution withwater. Subsequently the pH is adjusted to pH 4.5 with a 10% (w/w) NH₄OHsolution. The glucan content of the applied corn stover was 37% on drymatter.

The hydrolysis is done in a stirred, pH controlled and temperaturecontrolled reactor with a working volume of 1 l. Each hydrolysis isperformed in duplicate with 2.5 mg/g DM of TEC-210 cellulase enzymecomposition (or cocktail). TEC-210 was produced according to theinoculation and fermentation procedures described in WO2011/000949.

-   -   The following experiments are done:    -   1. 1 l of 20 % aCS, pH 4.5, temperature 62° C., stirrer speed 60        rpm, 3.5 mg TEC-210 cellulase composition per gram feedstock (on        dry matter), incubation time 120 hours (reference experiment) in        a closed reactor. The dissolved oxygen level of the reaction        mixture was measured constantly using a DO electrode. This slow        stirring resulted in a dissolved oxygen level 0.005 mol/m³.    -   2. As experiment 1 but using and enzyme dosage of 2.5 mg TEC-210        per gram feedstock (on dry matter) and a stirrer speed of 250        rpm a head space over the reaction mixture which is constantly        refreshed with fresh air. The higher stirring speed in        combination with refreshment of the head space with fresh air        resulted in a dissolved oxygen level of. 0.030 mol/m³ in the        reaction mixture.

During hydrolysis samples were taken for analysis. The samples werecooled on ice and immediately 50 μl of each supernatant is diluted in1450 μl grade l water. The diluted supernatant is subsequently filtered(0.45 μm filter, Pall PN 454) and the filtrates are analysed for sugarcontent as described below.

The sugar concentrations of the diluted samples are measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

The results are presented in FIG. 3.

The glucan conversions are listed in Table 2.

TABLE 2 glucan conversion levels. Enzyme dosage DO level Glucanconversion Experiment (mg/g dm) mol/m³ (%) 1 2.5 0.030 75 2 3.5 0.005 70

1. Process for preparing a sugar product from ligno-cellulosic material,comprising: a) optionally pre-treatment of the ligno-cellulosicmaterial; b) optionally washing of the optionally pre-treatedligno-cellulosic material; c) enzymatic hydrolysis of the optionallywashed and/or optionally pre-treated ligno-cellulosic material using anenzyme composition comprising at least two cellulase and whereby theenzyme composition at least comprises GH61; and d) optionally recoveryof a sugar product; wherein after the pre-treatment and before and/orduring the enzymatic hydrolysis oxygen is added to the ligno-cellulosicmaterial.
 2. Process for preparing a fermentation product fromligno-cellulosic material, comprising: a) optionally pre-treatment ofthe ligno-cellulosic material; b) optionally washing of the optionallypre-treated ligno-cellulosic material; c) enzymatic hydrolysis of theoptionally washed and/or optionally pre-treated ligno-cellulosicmaterial using an enzyme composition comprising at least two cellulaseand whereby the enzyme composition at least comprises GH61; d)fermentation of the hydrolysed ligno-cellulosic material to produce afermentation product; and e) optionally recovery of a fermentationproduct; wherein after the pre-treatment and before and/or during theenzymatic hydrolysis oxygen is added to the ligno-cellulosic material.3. Process according to claim 1, wherein during the enzymatic hydrolysisc) oxygen is added to the ligno-cellulosic material.
 4. Processaccording to claim 1, wherein the oxygen is added in the form ofbubbles.
 5. Process according to claim 1, wherein the reactor for theenzymatic hydrolysis has a volume of 1 m³ or more.
 6. Process accordingto claim 2, wherein the enzymatic hydrolysis time is 5 to 150 hours. 7.Process according to claim 1, wherein the enzyme composition usedretains activity for 30 hours or more.
 8. Process according to claim 1,wherein the hydrolysis is conducted at a temperature of 45° C. or more,optionally 50° C. or more and optionally at a temperature of 55° C. ormore.
 9. Process according to claim 1, wherein the enzyme composition isderived from a fungus, optionally microorganism of the genus Rasamsoniaor the enzyme composition comprises a fungal enzyme, a Rasamsoniaenzyme.
 10. Process according to claim 1, wherein the dry matter contentin the hydrolysis c) is 10 wt % or more, optionally is 14 wt % or moreand optionally is 14 to 33 wt %.
 11. Process according to claim 9,wherein the dry matter content in the hydrolysis c) is 14 to 33% wt %.12. A process according to claim 1 in which the enzymatic hydrolysistakes place in a batch, fed batch and/or continuous culture reactor. 13.A process according to claim 1 in which oxygen is introduced as anoxygen-containing gas optionally air.